A molecular portrait of the response to unfolded proteins
© GenomeBiology.com 2000
Published: 4 August 2000
Using DNA microarrays, 381 genes have been found to be induced in response to unfolded proteins. The identity of the previously characterized 208 of these, and further experiments, have revealed new details on the scope of the unfolded protein response and its connection to the degradation of proteins at the endoplasmic reticulum.
The endoplasmic reticulum (ER) serves as a way station for the transit of secreted proteins, and specifically as a depot devoted to the folding and maturation of proteins and to the assembly of macromolecular protein complexes. Before the protein passenger can exit the station, however, it is subject to a 'search' by the quality-control manager of the ER. The quality-control manager, sensing trouble, can retain undesirable passengers in the ER, and in certain cases banish them from the station. Undesirable passengers - aberrant polypeptides that cannot fold properly or mature - are sent back to the cytoplasm from which they arrived, and there they are degraded by the proteasome. This brutal treatment clears the ER of polypeptides that have the potential to aggregate, and prevents potentially toxic molecules from being secreted. The process by which aberrant, secreted polypeptides are degraded by the cytoplasmic proteasome is known as ER-associated degradation, or ERAD .
ER-associated protein degradation
The mechanism of ERAD has been uncovered in recent years through the analysis of ERAD-defective yeast mutants [2,3], through the development of in vitro assays in which ERAD can be measured , and through analyses of the fates of misfolded proteins in the secretory pathway [4,5,6,7,8]. Many disease-causing mutations in secreted polypeptides escape ERAD, and in some cases the over-zealous actions of the quality-control manager can promote disease - for example, in cystic fibrosis [9,10]. In addition, viruses may co-opt ERAD to destroy plasma-membrane-targeted host proteins and to elude the immune system, for example in infection by HIV  or cytomegalovirus . Bacterial toxins travel in reverse through the secretory pathway and finally exit from the ER, like ERAD substrates, to the cytoplasm .
The unfolded protein response
Foul conditions may increase the concentration of misfolded proteins in the ER and overwhelm the capacity of this way station. Unlike rail stations, though, overwhelmed eukaryotic cells simply increase the capacity of the ER to house the greater number of misfolded proteins, and synthesize molecular chaperones to help solubilize the denatured polypeptides. This unfolded protein response (UPR), a response to increased concentrations of misfolded polypeptides in the ER, is initiated by an ER-resident transmembrane protein, Ire1p [14,15], thought to sense the presence of aberrant proteins in the ER lumen. The cytoplasmic domain of this molecule, which has homology to both kinases and an RNase, then cleaves an intron from the primary transcript for the Hac1p transcription factor [16,17,18]. Ligation of the mRNA, which allows for subsequent translation, requires the tRNA ligase, Rlg1p . Once made, the transcription factor transits into the nucleus and initiates the synthesis of genes containing a 5' 'unfolded protein response element' and other UPR target genes (Figure 1) .
Although the identities of a few of the genes up-regulated by the UPR have been known for several years (for example, ER lumenal molecular chaperones), the full spectrum of proteins induced directly or indirectly by the UPR has been ill-defined. A recent paper in Cell from the Walter and Weissman laboratories  rectifies this problem, and provides fascinating details on the breadth of the UPR and the connection between the UPR and ERAD.
A protein profile of the UPR
To establish the complete profile of genes induced in response to unfolded proteins, yeast cells (Saccharomyces cerevisiae) were incubated with compounds known to compromise polypeptide folding in the ER: dithiothreitol (DTT), a reducing agent, and tunicamycin, an inhibitor of N-linked glycosylation. Yeast genomic oligonucleotide arrays were screened using biotinylated cRNA probes in collaboration with researchers from Affymetrix, Inc. [http://www.affymetrix.com]. The criterion for identifying UPR-target genes was that their transcription should not be stimulated in ire1 or hac1 mutant strains, but in wild-type cells transcription should be induced to levels similar to that of a group of seven previously identified UPR targets. One of these target genes, KAR2, encodes the lumenal Hsp70 molecular chaperone, BiP, which is known to play a vital role in protein translocation into the ER, protein folding in the ER, and ERAD [22,23,24,25]. Thus, Travers et al.  chose genes as UPR targets only if the level of their over-expression correlated to the canonical set at least as well as that observed for BiP. The ultimate result of this analysis yielded 381 open reading frames (ORFs). Some information on the function of the corresponding proteins is available for 208 of these, and no information is available for the other 173. One of the previously uncharacterized genes uncovered from the screen, PER100, is homologous to a gene required for protein translocation in a related yeast, Yarrowia lipolytica , and a per100 mutant was shown by Travers et al.  to exhibit ERAD defects. Clearly, continued analysis of the 173 previously uncharacterized ORFs will yield many exciting discoveries.
Links between the UPR and ERAD
About one half (103) of the previously characterized genes identified by Travers et al.  play roles in protein translocation, glycosylation, vesicular transport from the ER, protein targeting to the vacuole (which can also dispose of aberrant proteins in yeast), cell wall biosynthesis, and ERAD. In a related work, a screen to isolate yeast mutants that cannot survive in the absence of the UPR was performed, and the synthetic lethal mutants isolated from this screen uncovered genes similarly required for protein translocation, protein folding and glycosylation, and ERAD (D. Ng, personal communication). It is not difficult to imagine why these processes may be induced when the secretory way station is under siege.
Travers et al.  found further interactions between the UPR and ERAD. First, ERAD was less efficient in strains in which IRE1 was deleted. Second, induction of the UPR increased the efficiency of ERAD. Third, if the UPR, or the concentration of misfolded proteins, was elevated to very high levels by the introduction of DTT, tunicamycin, or an over-expression system for an ERAD substrate, ERAD was reduced. Taken together, these results indicate that the UPR helps reduce the mass of unfolded proteins in the ER via ERAD, but when this mass raises above a threshold level the efficiency of ERAD is compromised. Fourth, deletion of genes that are not essential for viability but are required for ERAD led to a modest (around twofold) induction of the UPR. And fifth, strains lacking one of these ERAD genes and IRE1 were nonviable at elevated temperatures, indicating that cells survive either a certain level of aberrant proteins in the ER or an inability to respond to unfolded proteins, but they succumb when the two are combined.
- McCracken AA, Brodsky JL: Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. J Cell Biol. 1996, 132: 291-298.PubMedView ArticleGoogle Scholar
- Knop M, Finger A, Braun T, Hellmuth K, Wolf DH: Derl, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 1996, 15: 753-763.PubMedPubMed CentralGoogle Scholar
- Hampton RY, Gardner RG, Rine J: Role of the 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell . 1996, 7: 2029-2044.PubMedPubMed CentralView ArticleGoogle Scholar
- Hiller MM, Finger A, Schweiger M, Wolf DH: ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science. 1996, 273: 1725-1728.PubMedView ArticleGoogle Scholar
- Biederer T, Volkwein C, Sommer T: Role of Cuel in ubiquitination and degradation at the ER surface. Science. 1997, 278: : 1806-1809. 10.1126/science.278.5344.1806.View ArticleGoogle Scholar
- Dürr G, Strayle J, Plemper R, Elbs S, Klee SK, Catty P, Wolf DH, Rudolph HK: The medial-Golgi ion pump Pmrl supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell. 1998, 9: 1149-1162.PubMedPubMed CentralView ArticleGoogle Scholar
- Werner ED, Brodsky JL, McCracken AA: Proteasome-dependent ER-associated protein degradation: an unconventional route to a familiar fate. Proc Natl Acad Sci USA. 1996, 93: 13797-13801. 10.1073/pnas.93.24.13797.PubMedPubMed CentralView ArticleGoogle Scholar
- Plemper RK, Egner R, Kuchler K, Wolf DH: Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome. J Biol Chem. 1998, 273: 32848-32856. 10.1074/jbc.273.49.32848.PubMedView ArticleGoogle Scholar
- Ward CL, Omura S, Kopito RR: Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995, 83: 121-127.PubMedView ArticleGoogle Scholar
- Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR: Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell. 1995, 83: 129-135.PubMedView ArticleGoogle Scholar
- Schubert U, Antón LC, Co JH, Bour S, Bennink JR, Orlowski M, Strebel K, Yewdell JW: CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway. J Virol. 1998, 72: 2280-2288.PubMedPubMed CentralGoogle Scholar
- Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, Ploegh HL: The human cytomegalovirus USII gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 1996, 84: 769-779.PubMedView ArticleGoogle Scholar
- Schmitz A, Herrgen H, Winkeler A, Herzog V: Cholera toxin is exported from microsomes by the Sec61p complex. J Cell Biol. 2000, 148: 1203-1212. 10.1083/jcb.148.6.1203.PubMedPubMed CentralView ArticleGoogle Scholar
- Mori K, Ma W, Gething M, Sambrook J: A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 1993, 74: 743-756.PubMedView ArticleGoogle Scholar
- Cox JS, Shamu CE, Walter P: Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993, 73: 1197-1206.PubMedView ArticleGoogle Scholar
- Mori K, Kawahara T, Yoshida H, Yanagi H, Yura T: Signaling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells. 1996, 1: 803-817. 10.1046/j.1365-2443.1996.d01-274.x.PubMedView ArticleGoogle Scholar
- Mori K, Ogawa N, Kawahara T, Yanage H, Yura T: mRNA splicing-mediated C-terminal replacement of transcription actor Haclp is required for efficient activation of the unfolded protein response. Proc Natl Acad Sci USA. 2000, 97: 4660-4665. 10.1073/pnas.050010197.PubMedPubMed CentralView ArticleGoogle Scholar
- Sidrauski C, Walter P: The transmembrane kinase Irelp is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell. 1997, 90: 1031-1039.PubMedView ArticleGoogle Scholar
- Sidrauski C, Cox JS, Walter P: tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell. 1996, 87: 405-413.PubMedView ArticleGoogle Scholar
- Chapman R, Sidrauski C, Walter P: Intracellular signaling from the endoplasmic reticulum to the nucleus. Annu Rev Cell Dev Biol . 1998, 14: 459-485. 10.1146/annurev.cellbio.14.1.459.PubMedView ArticleGoogle Scholar
- Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P: Functional and genomic analyses reveal essential coordination between the unfolded protein response and endoplasmic reticulum-associated degradation. Cell. 2000, 101: 249-258.PubMedView ArticleGoogle Scholar
- Brodsky JL, Goeckeler J, Schekman R: BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum. Proc Natl Acad Sci USA. 1995, 92: 9643-9646.PubMedPubMed CentralView ArticleGoogle Scholar
- Simons JF, Ferro-Novick S, Rose MD, Helenius A: BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast. J Cell Biol. 1995, 130: 41-49.PubMedView ArticleGoogle Scholar
- Plemper RK, Bohmler S, Bordallo J, Sommer T, Wolf DH: Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature. 1997, 388: 891-895. 10.1038/42276.PubMedView ArticleGoogle Scholar
- Brodsky JL, Werner ED, Dubas ME, Goeckeler JL, Kruse KB, McCracken AA: The requirement for molecular chaperones during ER-associated protein degradation (ERAD) demonstrates that protein import and export are mechanistically distinct. J Biol Chem. 1999, 274: 3453-3460. 10.1074/jbc.274.6.3453.PubMedView ArticleGoogle Scholar
- Boisrame A, Kabani M, Beckerich JM, Hartmann E, Gaillardin C: Interaction of Kar2p and Slslp is required for efficient co-translational translocation of secreted proteins in the yeastYarrowia lipolytica. J Biol Chem. 1998, 273: 30903-30908. 10.1074/jbc.273.47.30903.PubMedView ArticleGoogle Scholar